[1]陈萌萌,仇汝龙,范志宇,等.兔出血症病毒2型TaqMan探针荧光定量RT-PCR检测方法的建立及应用[J].江苏农业学报,2021,(06):1476-1480.[doi:doi:10.3969/j.issn.1000-4440.2021.05.015]
　CHEN Meng-meng,QIU Ru-long,FAN Zhi-yu,et al.Establishment and application of a TaqMan-based fluorescence quantitative real-time PCR assay for detection of rabbit hemorrhagic disease virus type 2[J].,2021,(06):1476-1480.[doi:doi:10.3969/j.issn.1000-4440.2021.05.015]
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兔出血症病毒2型TaqMan探针荧光定量RT-PCR检测方法的建立及应用()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2021年06期
页码:
1476-1480
栏目:
畜牧兽医·水产养殖
出版日期:
2021-12-30

文章信息/Info

Title:
Establishment and application of a TaqMan-based fluorescence quantitative real-time PCR assay for detection of rabbit hemorrhagic disease virus type 2
作者:
陈萌萌; 仇汝龙; 范志宇; 胡波; 宋艳华; 魏后军; 朱伟峰; 徐为中; 王芳
(江苏省农业科学院兽医研究所/农业部兽用生物制品工程重点实验室，江苏南京210014)
Author(s):
CHEN Meng-meng; QIU Ru-long; FAN Zhi-yu; HU Bo; SONG Yan-hua; WEI Hou-jun; ZHU Wei-feng; XU Wei-zhong; WANG Fang
(Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences/Key Laboratory for Veterinary Bio-Product Engineering, Ministry of Agriculture, Nanjing 210014， China)
关键词:
兔出血症病毒2型; TaqMan探针; 荧光定量RT-PCR; 敏感性; 特异性
Keywords:
rabbit hemorrhagic disease virus type 2; TaqMan probe; fluorescence quantitative real-time PCR; sensitivity; specificity
分类号:
S855.3
DOI:
doi:10.3969/j.issn.1000-4440.2021.05.015
文献标志码:
A
摘要:
以新发生的兔出血症病毒2型（RHDV2）SC 2020/04株VP60基因序列为参考，设计出1对特异性引物和TaqMan探针，建立了一种快速、灵敏且特异的用于检测新型病毒的荧光定量RT-PCR检测方法。试验结果显示：本研究建立的方法，标准曲线线性关系良好，R2值达到0.999；其特异性较好，与兔出血症病毒1型（RHDV1）、仙台病毒（SV）和轮状病毒（RRV）病原均无交叉反应；灵敏性高，最低检出量为1μl 1×10 拷贝；且重复性良好，批内和批间试验的变异系数平均值均小于2%。临床检测结果表明，利用该方法对108份临床病料进行检测，检出RHDV2阳性样品72份，检出率为66.7%，明显高于常规的RT-PCR方法(62.0%)。结果证明，新建立的TaqMan荧光定量RT-PCR方法适合于RHDV2感染的特异性诊断。
Abstract:
A rapid, sensitive and specific fluorescence quantitative real-time PCR (RT-qPCR) method for the detection of rabbit hemorrhagic disease virus type 2 (RHDV2) was established. A pair of specific primers and TaqMan probes were designed in the conserved region of VP60 gene of the new variant strain SC 2020/04. The results showed that the standard curve had a good linear relationship, the R2 was 0.999. The TaqMan-based RT-qPCR method had good specificity and no cross reactions with rabbit hemorrhagic disease virus type1 (RHDV1), sendai virus (SV) and rotavirus (RRV). The method established in this study had high sensitivity, and the minimum detectable amount was 10 copies per microlitre. Moreover, the repeatability was good, and the mean coefficient of variation was less than 2%. The TaqMan-based RT-qPCR method was used to detect 108 clinical samples. The detection rate was 66.7%, which was higher than that of conventional RT-PCR. To sum up，the newly established TaqMan-based RT-qPCR method is suitable for the specific diagnosis of RHDV2 infection.

参考文献/References:

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备注/Memo

备注/Memo:
收稿日期：2020-12-28基金项目：国家自然科学基金项目(31702274)；现代农业产业技术体系建设专项资金项目（CARS-43-C-1）作者简介：陈萌萌 (1987-) ，女，连云港人，博士，助理研究员，主要从事家兔疾病防治与兽医生物技术研究。(E-mail)moonchen2010@yeah.net通讯作者：王芳，(E-mail)rwangfang@126.com
更新日期/Last Update: 2022-01-07